Breast tumors with elevated expression of 1q candidate genes confer poor clinical outcome and sensitivity to Ras/PI3K inhibition.

Genomic aberrations are common in cancers and the long arm of chromosome 1 is known for its frequent amplifications in breast cancer. However, the key candidate genes of 1q, and their contribution in breast cancer pathogenesis remain unexplored. We have analyzed the gene expression profiles of 1635 breast tumor samples using meta-analysis based approach and identified clinically significant candidates from chromosome 1q. Seven candidate genes including exonuclease 1 (EXO1) are consistently over expressed in breast tumors, specifically in high grade and aggressive breast tumors with poor clinical outcome. We derived a EXO1 co-expression module from the mRNA profiles of breast tumors which comprises 1q candidate genes and their co-expressed genes. By integrative functional genomics investigation, we identified the involvement of EGFR, RAS, PI3K / AKT, MYC, E2F signaling in the regulation of these selected 1q genes in breast tumors and breast cancer cell lines. Expression of EXO1 module was found as indicative of elevated cell proliferation, genomic instability, activated RAS/AKT/MYC/E2F1 signaling pathways and loss of p53 activity in breast tumors. mRNA-drug connectivity analysis indicates inhibition of RAS/PI3K as a possible targeted therapeutic approach for the patients with activated EXO1 module in breast tumors. Thus, we identified seven 1q candidate genes strongly associated with the poor survival of breast cancer patients and identified the possibility of targeting them with EGFR/RAS/PI3K inhibitors

<i>EXO1</i> modular genes are tightly co-expressed and associated with poor prognosis in breast cancer patients

<p>: (A) Identification of <i>EXO1</i> modular genes using meta-analysis of correlation coefficients from multiple breast tumor datasets. Schematic <i>EXO1</i> centred network shows that <i>EXO1</i> co-expressed genes are involved in cell cycle and DNA repair. (B) and (C) Elevated expression of <i>EXO1</i> modular genes was associated with poor clinical outcome in breast tumor cohorts. (D) Heatmap showing the expression pattern of <i>EXO1</i> modular genes in 51 breast cancer cell lines. <i>EXO1</i> modular genes are highly expressed in aggressive and basal cell lines. I-Invasive, N-Non-invasive, B-Basal, L-Luminal and ER status of the cell lines are shown on the top.</p

Identification of signaling processes associated with <i>EXO1</i> expression in breast tumors.

<p>(A) The activation status of oncogenic signaling pathways and other cancer signatures in 198 breast tumor samples shown as heatmap. Different pathway or cellular process specific gene-sets were analyzed and the comprehensive activation (red) and inactivation (green) pattern across samples is shown. (B) Principal component analysis of the pathway activation pattern in breast tumors shown in Figure A. (C) Occurrence of E2F, Myc and p53 transcription factor binding sites in <i>EXO1</i> promoter. (D) Western blot showing the expression of <i>EXO1</i> in a panel of breast cancer cell lines. (E) RT-PCR showing the expression of <i>EXO1</i> in a panel of breast cancer cell lines (top panel). <i>In </i><i>vitro</i> pathway specific reporter assays showing relative activation of Myc and E2F transcription factors in three of the higher level <i>EXO1</i> expressing cell lines (ER –ve) while compared to two lower level <i>EXO1</i> expressing cell lines (ER +ve) (Lower panel). These assays have been performed thrice with triplicates. (F) Luciferase reporter assay showing the activation of <i>EXO1</i> promoter upon the transfection of E2F1 or c-Myc cDNA in MDA-MB231 cells (p-values were 0.013 and 0.009 respectively). pGL3-<i>EXO1</i> is <i>EXO1</i> promoter construct in pGL3 enhancer reporter vector. The experiment has been performed thrice with triplicates.</p

Identification of genes associated with poor clinical outcome in 1q amplicon of breast tumors.

<p>(A) High frequency amplification of 1q region in multiple cohorts of breast tumors. Graphs I – VI represent inferred copy-number of probes at chromosome 1 from the aCGH studies [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077553#B61" target="_blank">61</a>-<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077553#B66" target="_blank">66</a>]. These graphical representations were the outcome of data visualized through Progenetix [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077553#B67" target="_blank">67</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077553#B68" target="_blank">68</a>]. Copy number gain and loss are represented with green and red colors respectively. In all these five cohorts, 1q is amplified in 40 - 60 % of tumors. (B) Meta analysis – workflow for the identification 1q genes associated with patient survival. (C) Cox regression coefficient, hazard ratio and p-value of the shortlisted 1q genes, (D) Chromosomal map showing the locations of clinically significant 1q genes. (E) The identified 1q candidate genes showing an elevated expression in breast tumors when compared to the non-cancerous breast tissues in 3 different cohorts. </p

Ras/PI3K inhibition is the suitable therapeutic approach for breast tumors with elevated <i>EXO1</i> modular expression.

<p>(A) Workflow employed for the identification of suitable chemo therapeutic agent for <i>EXO1</i> module expressing breast tumors. (B) Results of Connectivity map analysis showing that while patients expressing <i>EXO1</i> co-regulated genes, Pi3K inhibition could be the possible targeted therapeutic approach. (C) Heatmap showing the inhibition of <i>EXO1</i> modular genes in GSE33403 downloaded from GEO that corresponds to MCF10A cells (PIK3CA E545K mutation) treated with LY294002 for 4 hours and 24 hours. Similarly, the profiles of A549 and SHEP cells treated with the Ras inhibitor FTS were obtained from Blum et al., 2007. (D) RT-PCR showed reduced expression of 5 out of 7, 1q candidate genes <i>NEK2, CKS1B, DTL, KIF14</i> and <i>EXO1</i> in MB231 cells upon chemical inhibition of RAS pathway with salirasib.</p

Schematic representation of signaling pathways/factors possibly regulating 1q candidate gene expression in breast cancer.

<p>EGFR, RAS, PI3K / AKT, MYC, and E2F were identified as the upstream regulators of these genes. The sequential arrangement of pathways is from the well established literature [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077553#B49" target="_blank">49</a>-<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077553#B51" target="_blank">51</a>]. Apart from the pathways, genomic instability, telomerase activation and loss of p53 are also associated with the expression of 1q candidate genes.</p